Failure of the ubiquitin proteasome machinery and molecular chaperones may result in aggregation of mutant or damaged polypeptides leading to various devastating diseases. However, special molecular machinery has evolved as a last line of defense to relieve proteotoxicity by transporting misfolded protein aggregates/oligomers to the centrosome-localized aggresome. The main objective of this proposal is to uncover the mechanisms of aggresome formation. The proposed work is based on our yeast model of aggregation of proteins with expanded polyglutamine (polyQ) domains. In the past, this model was used to identify small molecules that suppress polyQ aggregation, improve motor performance and reduce neuronal atrophy in a mouse model of HD. With this model, we have established that (a) aggresome formation both in yeast and mammalian cells requires transferable aggresome-targeting signals on substrate proteins, e.g. the proline-rich region (P-region) of exon 1 of huntingtin, (b) an SH3-domain protein Boi2 serves as a recognition element for the P-region aggresome- targeting signal, (c) a yeast 14-3-3 protein Bmh1 and components of the Cdc48/VCP-Ufd1-Nlp4 complex play an essential role in aggresome formation. We also established a cell culture model of aggresome formation to investigate the relevance for mammalian cells of aggresome components identified in yeast. In Aim 1 we will clarify how the aggresome machinery recognizes small polyQ aggregates/oligomers. We will establish the role of the SH3-domain protein Boi2 in the recognition of the aggregates in yeast, and will clarify whether Boi2 homologs function in mammalian cells to recognize huntingtin and other polyQ-containing pathological proteins. In Aim 2 we will establish the function of the 14-3-3 protein Bmh1 in early stages of aggresome formation in yeast, and will test whether 14-3-3 proteins play a general role in aggresome formation in mammalian cells. These experiments will help to clarify how the aggresome machinery distinguishes small aggregates/oligomers of abnormal polypeptides from soluble monomers. In Aim 3 using both biochemical and genetic approaches we will identify novel components involved in aggresome formation. As a result of this work, we plan to obtain sufficient information for understanding the mechanism of aggresome formation. PUBLIC HEALTH RELEVANCE Many devastating diseases, including major neurodegenerative disorders, are caused by accumulation of abnormal proteins. These abnormal species tend to aggregate, and here we will establish cellular mechanisms of protein aggregation. This work will uncover how organisms try to protect themselves from development of Hungtington's and certain other diseases.